Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area following ischemic brain injury.In this study,an apoptotic model in rat hippocampal neurons was established by 0.5 mmol/L 3-morpholinosyndnomine(SIN-1),a nitric oxide donor.The models were then cultured with 0.1 mmol/L of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(DIDS;the chloride channel blocker) for 18 hours.Neuronal survival was detected using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay,and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining.Western blot analysis and immunochemiluminescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins.Real-time PCR was used to detect the mRNA expression of CIC-3.The results showed that SIN-1 reduced the neuronal survival rate,induced neuronal apoptosis,and promoted ClC-3 chloride channel protein and mRNA expression in the apoptotic neurons.DIDS reversed the effect of SIN-1.Our findings indicate that the increased activities of the ClC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.
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